PROJECT SUMMARY/ABSTRACT Inflammatory bowel disease (IBD) is an idiopathic, chronic, progressive, relapsing, and immunologically mediated condition. In spite of remarkable advances in our understanding of the clinical manifestation, epidemiology and risk factors, challenges remain in identifying the pathogenesis of IBD. Advances over the past four decades have led to the understanding that IBD is a complex and multifactorial disease that might emerge from interaction and integration of multiple components, including genetic susceptibility, microbiota imbalance, epithelial barrier dysfunction, and aberrant immune response. Epidemiological studies found that early-life infection is a risk factor for the development of pediatric and adult IBD, and gastrointestinal infection in adolescents and adults is a trigger for the onset or exacerbation of IBD. However, it is not clear whether and how these two events cause an aggravated and prolonged intestinal barrier dysfunction, which is the hallmark of IBD. Neonatal life is a uniquely vulnerable period during which neonates are susceptible to environmental challenges. Some of those challenges can induce durable epigenetic changes, predisposing the host to complex diseases that occur many decades after those initial events. We and others have shown that severe neonatal inflammation (NI) permanently alters gene expression in the colon and stomach, leading to motility dysfunction and functional dyspepsia-like symptoms, in a rat model of NI induced by trinitrobenzene sulfonic acid (TNBS), which provides a valuable preclinical model for research on disease susceptibility. Our preliminary findings suggest that NI causes stable reduction of butyrate-producing bacteria in the gut, resulting in heightened colonic epithelial permeability when subjected to a second inflammatory insult in adult life. Our central hypothesis is that neonatal colonic inflammation renders the host susceptible to intestinal barrier dysfunction by inducing functional microbiota imbalances that epigenetically target MMP9E-cadherin interactions. The hypothesis will be tested through two specific aims. In Aim 1, by using a newly-established two-hit rat model, we seek to demonstrate that NI alters composition of gut microbiome, production of short chain fatty acid, and expression of MMP9 and E- cadherin. Together, these changes induce susceptibility to IBD-like aggravated intestinal barrier dysfunction and abnormal immune response, which should be mitigated by butyrate supplementation. We will also validate the neonatal injury findings in IL10 deficient mice, an established IBD model. In Aim 2, by using human enteroids we will test the hypothesis that NI induces demethylation of DNA and histones at the MMP9 promoter, making it more susceptible to histone hyperacetylation and activation, when exposed to proinflammatory stimuli. Our results should help establish a novel paradigm that NI induces long-lasting epigenetic modifications to increase susceptibility to IBD. Our mechanistic findings on NI-induced epigenetic modifications, MMP9 activation, and E- cadherin cleavage should also provide new insights important for the design of preventive and therapeutic strategies for IBD.